Multivariate Study of the Effects of Combined Cinnamon, Clove, and Lemongrass Essential Oils on Enhancing Antioxidant and Antimicrobial Activity

Multivariate Study of the Effects of Combined Cinnamon, Clove, and Lemongrass Essential Oils on Enhancing Antioxidant and Antimicrobial Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Multivariate Study of the Effects of Combined Cinnamon, Clove, and Lemongrass Essential Oils on Enhancing Antioxidant and Antimicrobial Activity Thamyres César Albuquerque Sousa, Thaís Regina Rodrigues Vieira, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8041198/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A multivariate mixture system of EOs was employed to evaluate individual, binary, and tertiary effects of cinnamon, clove, and lemongrass essential oils on in vitro antioxidant activity and antimicrobial activity against Botrytis cinerea , Escherichia coli , and Staphylococcus aureus . The highest antioxidant activity was observed with 100% clove oil, showing inhibition rates of 94.97% (DPPH), 93.85% (ABTS). The addition of clove EO to the mixture, in a two-thirds proportion, resulted in inhibition percentages above 80% for both DPPH and ABTS. Regarding the inhibitory effect Botrytis cinerea , lemongrass EO exhibited the best performance, with 68% inhibition, while its combination, even partially, with clove and cinnamon produced a negative effect. Escherichia coli and Staphylococcus aureus , the strongest inhibitory activity was obtained with 100% lemongrass EO (inhibition zones of 7.1 and 6.7 cm). The simultaneous use lemongrass and cinnamon EOs in higher proportions within the mixture resulted in greater inhibition, suggesting a synergistic effect. Botrytis cinerea Bioactive Compounds Escherichia coli Mixture Design Staphylococcus aureus Figures Figure 1 Figure 2 1. Introduction Essential oils (EOs) are natural substances produced by plants that exhibit a wide range of bioactivities, including antioxidant, antimicrobial, antitumor, anti-acetylcholinesterase, and anti-α-glucosidase activities, among others. These biological properties are mainly attributed to the presence of terpenes, terpenoids, phenols, aldehydes, ethers, and other organic compounds (Krishnan et al., 2023 ; Niu et al., 2024 ; Caputo et al., 2022 ; You et al., 2022 ). EOs have been increasingly produced for use in aromatherapy, culinary applications, as food additives, flavor enhancers, fragrances, and other anthropogenic purposes (Sharifi-Rad et al., 2017 ). Lemongrass EO ( Cymbopogon citratus ) exhibits antioxidant, antifungal, and antimicrobial activities, making it highly promising for applications aimed at extending food shelf life (Akakpo et al., 2023 ). When applied to tomatoes stored at room temperature (25°C) and under refrigeration (8°C) infected with Colletotrichum gloeosporioides , lemongrass EO reduced the incidence (up to 63%) and severity (up to 9%) of anthracnose at room temperature (within 7 days) and completely inhibited the disease (0% incidence and severity) under refrigeration (within 40 days (Flores & Poveda, 2025 ). Clove EO ( Syzygium aromaticum ) has also demonstrated antioxidant and antimicrobial properties in various studies, proving effective and easy to apply (Pandey et al., 2023 ; Suttiarporn et al., 2024 ). Cinnamon EO is widely used in the food flavoring and cosmetics industries and has shown significant inhibitory effects on most foodborne microorganisms. As a preservative, it has been reported to delay the spoilage of a wide range of foods such as lemons, bread, strawberries, and cheeses (Zhang et al., 2023 ). In a study conducted by Albuquerque and collaborators, clove, cinnamon, and lemongrass EOs exhibited strong antifungal activity against Botrytis cinerea , with minimum inhibitory concentrations of 0.85 mg mL⁻¹, highlighting their potential to combat this pathogen (Albuquerque Sousa et al., 2024 ). Botrytis cinerea , he causal agent of gray mold, is one of the most notorious pathogens affecting fruits such as strawberries, leading to severe economic losses both in field production and during the postharvest period. Its impact is largely due to its distinct characteristics, including a short life cycle, massive reproductive capacity, and high genetic variability (Yousef et al., 2024 ). This fungus is considered a major contributor to crop losses, with estimates indicating that it is responsible for at least 30% of global production loss annually. It can infect more than 1,400 plant species across nearly 600 genera worldwide, resulting in staggering annual production losses estimated between USD 10 and 100 billion (Ullah et al., 2024 ). Essential oils play an important role in ensuring food safety against microorganisms such as fungi and bacteria, in addition to extending shelf life through their strong antioxidant potential. Food safety is a critical concern for public health, as foodborne diseases affect millions of people worldwide, causing significant economic losses and various health problems (Anamul Hasan Chowdhury et al., 2024 ). Among the pathogens most commonly associated with foodborne illnesses, Escherichia coli and Staphylococcus aureus are among the most prevalent microorganisms. Escherichia coli is a pathogenic microorganism that contaminates food globally, contributing to 600 million foodborne illnesses and 420,000 deaths annually resulting from the consumption of contaminated food (Jangid et al., 2024 ). Its main symptoms in humans include stomach discomfort, diarrhea, and vomitin (Amente et al., 2022 ). Staphylococcus aureus is a bacterium that produces staphylococcal enterotoxins, whose presence in food can cause outbreaks of food poisoning. This bacterium is part of the microbiota of animals and can spread to food products, with humans also serving as reservoirs (Duchez et al., 2025 ). According to statistics from the National Healthcare Safety Network (NHSN) surveillance system and the Emerging Infections Program (EPS), Staphylococcus aureus infections have been associated with approximately 50,000 annual deaths in the United States. Outbreaks have also been reported in South Korea; between 2002 and 2012, 2,357 cases from 174 outbreaks of staphylococcal food poisoning were documented (Kim et al., 2025 ). Several studies have demonstrated the effectiveness of EOs when applied individually. Research with lemon leaf EO ( Citrus limon ) revealed antioxidant activity against the DPPH radical, with an IC50 of 10.63 ppm, and a minimum inhibitory concentration against Staphylococcus aureus of 7.8 µg mL⁻¹. Willow leaf EO ( Hygrophila salicifolia ) exhibited antioxidant activity against the DPPH radical at 2.13 mg mL⁻¹ (Xu et al., 2024 ; Yeasmin et al., 2024 ). Multiple studies have shown that EOs possess antifungal effects, including activity against the pathogen B. cinerea . EO from Salvia officinalis (Rguez et al., 2019 ) and EO from Cupressus sempervirens (Rguez et al., 2018 ) demonstrated efficient in vitro antifungal activity against Botrytis cinerea , while EO from Tetraclinis articulata , applied at a dose of 100 µg mL⁻¹ on tomato fruits, reduced infection caused by Botrytis cinerea by 64.01%(Rguez et al., 2020 ). Synergism or antagonism between bioactive compounds remains a developing topic in research (Arora et al., 2023 ). Clove and lemongrass essential oils, when used in combination, demonstrated reduced minimum inhibitory concentration (MIC) values against the fungal phytopathogen Fusarium oxysporum f. sp. lycopersici , exhibiting synergism (Sharma et al., 2018 ). However, scientific evidence regarding the synergistic or antagonistic effects of lemongrass, cinnamon, and clove essential oils on antioxidant, antimicrobial, and antifungal activities (particularly the inhibition of Botrytis cinerea ) is still limited to our knowledge. If the concentrations of each oil are optimized, the use of essential oil blends may reduce the required amounts of active compounds, enhancing their effects with lower concentrations of each oil while minimizing potential sensory and economic impacts. In this context, a multivariate mixture design is a straightforward statistical approach to efficiently optimize combinations, reducing the number of experiments while obtaining high-quality information. This method makes it possible to estimate interactions among variables and to construct predictive models that relate responses to variables (Zonfrillo et al., 2025 ). Identifying optimal experimental conditions is a complex task, and multivariate optimization is essential to establish mathematical models capable of predicting the best conditions that meet the selected responses (Effting et al., 2025 ). Therefore, the aim of this study was to employ a multivariate mixture optimization of lemongrass, clove, and cinnamon essential oils to investigate the interaction effects among these oils on antioxidant activity against DPPH and ABTS radicals and on antimicrobial activity against Botrytis cinerea , Escherichia coli , and Staphylococcus aureus. 2 Material and methods 2.1 Material and reagents The plant material for the extraction of cinnamon and clove essential oils was purchased from local commerce in Pelotas, Rio Grande do Sul, Brazil (coordinates 31° 46′ 19″ S, 52° 20′ 34″ O), while the plant material for the extraction of lemongrass essential oil was obtained from a rural producer in Capão do Leão, Rio Grande do Sul, Brazil (coordinates 31°45'48"S e 52°29'02"O). The culture media used were potato dextrose agar (PDA), obtained from KASVI (Spain); tryptic soy broth (TSB), KASVI (Spain); eosin methylene blue agar (EMB), KASVI (Spain); Baird-Parker agar, NutriSelect Plus (Germany); and Mueller-Hinton agar, KASVI (Spain). 2,2-diphenyl-1-picrylhydrazyl (DPPH), CAS 1898-66-4, and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), CAS 30931-67-0, were purchased from Sigma-Aldrich (Germany). Methanol PA, CAS 67-56-1, and potassium persulfate, CAS 7727-21-1, were obtained from Êxodo Científica (Brazil). The fungi were isolated from strawberry plants ( Fragaria × ananassa Duch.) cultivated in the region of Pelotas, Brazil, and identified by their morphological characteristics (grayish growth, branched hyphae and conidiophores, unicellular ovoid conidia, colorless or grayish at the apex of conidiophores) as Botrytis cinerea . Standard bacterial strains of Escherichia coli (ATCC 43895) and Staphylococcus aureus were used (ATCC 10832). 2.2 Extraction of essential oils The extraction of essential oils was carried out by hydrodistillation using a fixed Clevenger apparatus, according to the method described by Albuquerque Sousa et al., ( 2024 ). 1. Multivariate mixture design The design employed was of the Simplex Centroid type, as described by Neto (2010). In this system, the experimental points are symmetrically distributed, making it possible to study the individual effects of the oils, as well as the binary and ternary combination effects. Table 1 presents the variables and levels studied, which resulted in the evaluation of 10 mixture combinations, each performed in triplicate and in random order. Table 1 – Variables, levels, and responses of the multivariate mixture design Mixture Composition Antioxidant activity (% inhibition) Antimicrobial activity Cinnamon Clove Lemongrass DPPH ABTS B. cinerea (% inhibition) E. coli (cm of halo) S. aureus (cm of halo) 6 0.5 0 0.5 16.6 17.29 36.25 5.25 6.40 6 0.5 0 0.5 5.4 10 37.50 5.85 6.40 6 0.5 0 0.5 2.14 9.86 36.25 5.00 7.20 7 0.33 0.33 0.33 91.14 76.14 46.67 4.25 6.85 7 0.33 0.33 0.33 91.96 79.57 47.50 5.25 6.50 7 0.33 0.33 0.33 91.96 79.71 45.42 5.00 5.40 8 0.67 0.17 0.17 86.66 70.43 31.25 6.50 3.95 8 0.67 0.17 0.17 86.56 71.86 30.00 6.30 4.40 8 0.67 0.17 0.17 86.66 71.43 29.17 6.85 4.15 9 0.17 0.67 0.17 94.6 82.57 36.25 4.50 5.50 9 0.17 0.67 0.17 94.7 83.57 33.33 4.10 6.00 9 0.17 0.67 0.17 95.11 85.57 31.25 4.65 5.65 10 0.17 0.17 0.67 91.75 63.71 45.42 6.40 5.50 10 0.17 0.17 0.67 92.06 64.14 45.83 5.50 6.30 10 0.17 0.17 0.67 91.24 62.86 42.50 6.15 5.75 Higher values are represented in the green scale, intermediate values in yellow, and lower values in the red scale For the analyses of antioxidant activity and antifungal effect against Botrytis cinerea , the three pure EOs were diluted to a concentration of 0.08 mg mL⁻¹ in 1X phosphate-buffered saline (PBS, pH 7.4). A total of 500 mL of PBS solution was prepared containing 4 g of sodium chloride, 0.1 g of potassium chloride, 0.72 g of disodium phosphate, and 0.1225 g of monopotassium phosphate in distilled water. This concentration was defined based on a previous study by the research group, which demonstrated inhibitory effects against the fungus(Albuquerque Sousa et al., 2024 ). hus, the mixtures were prepared with the following composition (in mg/mL): 1 – Cinnamon EO 0.08; 2 – Clove EO 0.08; 3 – Lemongrass EO 0.08; 4 – Cinnamon EO 0.04 + Clove EO 0.04; 5 – Clove EO 0.04 + Lemongrass EO 0.04; 6 – Cinnamon EO 0.04 + Lemongrass EO 0.04; 7 – Cinnamon EO 0.0267 + Clove EO 0.0267 + Lemongrass EO 0.0266; 8 – Cinnamon EO 0.053 + Clove EO 0.0133 + Lemongrass EO 0.0133; 9 – Cinnamon EO 0.0133 + Clove EO 0.053 + Lemongrass EO 0.0133; 10 – Cinnamon EO 0.0133 + Clove EO 0.0133 + Lemongrass EO 0.053. For the analyses of antibacterial activity against Escherichia coli and Staphylococcus aureus , each pure EO was diluted to a concentration of 1 mg mL⁻¹. Thus, the mixtures had the following composition (in mg/mL): 1 – Cinnamon EO 1.00; 2 – Clove EO 1.00; 3 – Lemongrass EO 1.00; 4 – Cinnamon EO 0.50 + Clove EO 0.50; 5 – Clove EO 0.50 + Lemongrass EO 0.50; 6 – Cinnamon EO 0.50 + Lemongrass EO 0.50; 7 – Cinnamon EO 0.34 + Clove EO 0.33 + Lemongrass EO 0.34; 8 – Cinnamon EO 0.67 + Clove EO 0.16 + Lemongrass EO 0.16; 9 – Cinnamon EO 0.16 + Clove EO 0.67 + Lemongrass EO 0.16; 10 – Cinnamon EO 0.16 + Clove EO 0.16 + Lemongrass EO 0.67. The responses studied were antioxidant activity (measured by the percentage of inhibition of DPPH and ABTS radicals) and antimicrobial activity (measured by the inhibition zones of Botrytis cinerea , Escherichia coli , and Staphylococcus aureus strains). Subsequently, the responses obtained were evaluated for the possibility of developing mathematical models with adequate fit to explain the interaction effects and the behavior of EO mixtures in relation to the responses, as well as to predict new mixture combinations and their effects. The models were analyzed by means of Analysis of Variance (ANOVA) using Statistica 12 software (StatSoft, USA), with a 95% confidence level. 2. Methods for antioxidant and antifungal activity analysis The antioxidant capacity of the microparticles was evaluated based on their ability to donate hydrogen to the stable free radical DPPH (Rufino et al., 2007a) and to scavenge the free radical ABTS (Rufino et al., 2007b), with minor modifications. For the DPPH assay, 100 µL of the EO dilutions were added to 3.9 mL of a 0.06 mM methanolic DPPH solution. The reaction was carried out at 25°C, in the absence of light, for 1 h, and absorbance was measured at 515 nm. For the ABTS assay, the free radical was prepared by reacting 5.7 mL of a 7 mM ABTS solution with 0.1 mL of a 140 mmol/L potassium persulfate solution at 25°C, under low light, for 16 h. Then, 30 µL of the EO dilutions were added to 3 mL of ABTS solution, and absorbance was measured at 734 nm. The results were expressed as percentage inhibition according to the equation below: Inhibition (%) = (A control − A amostra )/A control * 100, where A represents absorbance. The antifungal activity against Botrytis cinerea was determined using the method described by Gakuubi et al. ( 2017 ), with some modifications. Initially, Botrytis cinerea was pre-cultivated at 25°C for 72 h on potato dextrose agar (PDA). Approximately 25 mL of sterile PDA were prepared and poured into sterile Petri dishes at a temperature below 50°C. Immediately afterward, the EO mixtures were added and homogenized in the still-liquid PDA medium. A well of approximately 6 mm in diameter was made at the center of the plate, where a PDA disk containing the cultivated fungus (also 6 mm in diameter) was placed. The plates were then sealed and incubated at 25°C for 96 h. The growth halos were measured daily during incubation to calculate the percentage of inhibition. A plate without the addition of EO mixtures was used as the control. 2.5 Analysis of antibacterial activity. The antibacterial analysis against Escherichia coli and Staphylococcus aureus was carried out according to the protocol proposed by the Clinical and Laboratory Standards Institute Manual – CLSI (CLSI, 2005), with modifications. For reactivation, an inoculum of each bacterium was transferred to tryptic soy broth (TSB) and incubated in an oven for 24 h at 37°C. Subsequently, an inoculum from this growth was streaked onto Petri dishes with selective media, eosin methylene blue agar (EMB) for Escherichia coli and Baird-Parker agar for Staphylococcus aureus , and incubated for 24 h at 37°C to isolate the colonies. From the bacterial growth on Petri dishes, an inoculum was taken and resuspended in saline solution (0.85% NaCl), which was standardized to a 0.5 concentration on the McFarland scale (1.5 × 10⁸ CFU mL⁻¹). For the well diffusion test, the saline solution containing the bacterial inoculum was spread with the aid of a sterile swab onto the surface of Muller-Hinton agar plates (for Staphylococcus aureus ) and EMB agar plates (for Escherichia coli ), where wells had been previously made. Then, 10 µL of the mixtures were placed into the wells, and the plates were incubated for 24 h at 37°C. After this period, the inhibition zones were measured, and the results were expressed in centimeters. 3 Results and discussions Table 1 presents the results obtained for antioxidant, antifungal, and antibacterial activities provided by the mixtures of the experimental design. Table 2 shows the validation of the mathematical models regarding lack of fit, significance of the regressions, and significant coefficients. Table 2 Validation of the mathematical models of the essential oil mixture design Responses Model Significant Model Coefficients (error) F Lack of Fit F critical F Model Significance F critical A B C AB AC BC ABC % DPPH Inhibition Special Cubic 86.75 162.13 178.99 733.29 150.70 F 5.18 = 2.77 27.28 F 6.23 = 2.53 (8.86) (44.53) (44.53) (294.31) % ABTS Inhibition Special Cubic 19.07 89.09 12.50 80.94 61.60 771.50 78.98 F 5.18 = 2.77 52.87 F 6.23 = 2.53 (5.05) (5.05) (5.05) (25.36) (25.36) (167.61) % Botrytis Inhibition Quadratic 47.79 45.23 68.72 -83.31 -73.78 51.2 F 6.18 = 2.66 15.64 F 5.24 = 2.62 (3.12) (3.12) (3.12) (14.34) (14.34) Inhibition zone E. coli Special Cubic 6.06 4.60 7.20 5.10 -3.71 21.44 7.25 F 5.18 = 2.77 7.93 F 6.23 = 2.53 (0.32) (0.32) (0.32) (1.63) (1.63) (10.75) Inhibition zone S. aureus Quadratic 4.64 5.98 6.63 -3.90 6.91 F 6.18 = 2.66 4.78 F 5.24 = 2.62 (0.37) (0.37) (0.37) (1.71) A: Cinnamon essential oil. B: Clove essential oil. C: Lemongrass essential oil; 3.1 Antioxidant activity For antioxidant activity, measured through the inhibition of DPPH and ABTS radicals, the model that best explained the behavior of the variables was the special cubic model. However, in the lack-of-fit test, it was observed that for DPPH the calculated F value was 150.70, far exceeding the F calculate value of 5.18 and 2.77. This high lack of fit makes the use of this mathematical model unfeasible for predicting optimal conditions. The same occurred for the ABTS model, with a calculated F value of 78.98, also exceeding the F calculate value of 5.18 and 2.77. Therefore, the interpretation of the observed effects for these responses was based on the experimental results (Table 1 ). When the essential oils were studied individually (mixtures 1, 2, and 3), the best antioxidant activity was observed in clove oil (mixture 2), with 94.97% inhibition for DPPH and 93.85% for ABTS. Pure cinnamon and lemongrass essential oils exhibited low antioxidant activity (below 9% for DPPH and 17% for ABTS). For the binary mixtures, whenever clove oil was present, inhibition was greater than 90% for DPPH and greater than 66% for ABTS, regardless of whether it was combined with cinnamon or lemongrass oil (mixtures 4 and 5). In the absence of clove (binary mixture of cinnamon and lemongrass), inhibition was lower than 18% for both radicals. For the ternary mixtures (mixtures 7, 8, 9, and 10), inhibition of the DPPH radical was greater than 86%, and inhibition of the ABTS radical was greater than 62%. The highest inhibition for the ABTS radical was observed in mixture 9, with values above 82%, which contained a higher proportion of clove essential oil. The presence of clove oil consistently resulted in the best antioxidant activity. When clove EO accounted for one-third of the mixture (mixture 7), the antioxidant activity achieved was above 90% for DPPH and above 76% for ABTS. This indicates the possibility of varying the combinations used depending on cost, commercial availability, and sensory compatibility. The biological activity of clove essential oil results from its high content of bioactive compounds such as eugenol, which contains a phenolic group of interest due to its strong antioxidant capacity, as well as antimicrobial activity, including action against foodborne pathogenic bacteria (El-Saber Batiha et al., 2020 ; Haro-González et al., 2021 ; Khalil et al., 2017 ; Ulanowska & Olas, 2021 ). Citral, the main constituent of lemongrass essential oil, is known for its anti-inflammatory, immunomodulatory, fungistatic antimicrobial, antioxidant, and antiseptic properties (Alagawany et al., 2021 ). Cinnamon essential oil has cinnamaldehyde as its major compound and is widely used in the pharmaceutical and food industries for its strong antioxidant and antifungal properties (Benmoussa et al., 2023 ). Regarding antioxidant activity, it can be observed that although the models showed a high lack of fit, the significant coefficients were consistent with the experimental results, including their numerical magnitude. One possible reason for a false outcome in the lack-of-fit test is when the relative standard deviation of the replicates is very low. However, the deviation in this study was within normal limits and did not affect the test. The levels were selected to allow testing of several combinations in different proportions. The lack of fit is possibly due to the absence of terms in the model to account for the variance (which would require a larger number of experiments) or to the range between the levels studied. 3.2 Antifungal activity For the activity against the fungus Botrytis cinerea , the model that best explained the behavior of the variables was the quadratic model. However, in the lack-of-fit test, the F calculated value was 51.20, which was much higher than the F critical 6,18 of 2,66. When interpreting the effects observed from the experimental results (Table 1 ), it can be noted that when the essential oils were tested individually (mixtures 1, 2, and 3), the strongest inhibitory effect against the fungus was obtained with lemongrass essential oil, with values above 67%, followed by cinnamon oil with values above 49% and clove oil with values above 44%. For the binary and ternary mixtures (mixtures 4, 5, 6, 7, 8, 9, and 10), all showed inhibition values lower than those of the oils when used alone, indicating that the oil mixtures did not produce a synergistic effect but rather an antagonistic one, reducing the inhibitory effect against the fungus due to the lower proportion of clove oil. Despite the lack of fit in the quadratic model, the coefficients also pointed to similar responses. The most significant mathematical effects were the negative interaction effects (AC and BC), namely between cinnamon essential oil (A) and lemongrass (C), and between clove essential oil (B) and lemongrass. The model also confirmed the experimental data, highlighting that the third most important effect was the positive contribution of lemongrass to the inhibition percentage when this EO was used individually (C). 3.3 Antibacterial activity For the antibacterial activity against the pathogens Escherichia coli and Staphylococcus aureus , the model that best explained the behavior of the variables was the special cubic model for Escherichia coli and the quadratic model for Staphylococcus aureus . In the lack-of-fit test, the F calculated value for Escherichia coli was 7.25, slightly higher than the F critical value of 5.18 and 2.77. For the Staphylococcus aureus model, the F calculated value was 6.91, slightly higher than the F critical 6.18 value and 2.66. Both models showed a slight lack of fit. However, since these values were less than 10 times the F critical value, several researchers have successfully employed them to predict optimal conditions. Therefore, both models were used for this purpose (Caroline Paz Gonçalves et al., 2024 ; Rosas et al., 2024 ). The significant coefficients presented in Table 2 are consistent with the values observed in the experiment (Table 1 ). For Escherichia coli, pure lemongrass EO (coefficient C) showed the largest inhibition zone (greater than 7 cm), corroborating the experimental results reported in Section 3. The next most significant effect was observed with cinnamon EO, followed by clove EO, both of which also had significant coefficients. Regarding binary effects, coefficient AB (relating cinnamon and clove, mixture 4 in the experiment) showed a positive interaction in terms of bacterial inhibition. On the other hand, coefficient AC (mixture 6) showed a negative interaction between cinnamon and lemongrass oils. The simultaneous use of the three EOs also showed a positive effect. When lemongrass EO was present in the mixture of the three EOs at a proportion of two-thirds (mixture 10), the inhibition zone was greater than 6 cm, a value higher than that obtained with cinnamon or clove oils when used alone. Figure 1 presents the response surface for the effect of the mixtures of clove, cinnamon, and lemongrass essential oils on the inhibition of Escherichia coli, showing that higher concentrations of lemongrass maximize inhibition, while mixtures with a predominant proportion of this oil also maintain high efficacy. For the inhibition of Staphylococcus aureus , the best antibacterial activity was observed with lemongrass essential oil (mixture 3), with an average inhibition zone of 6.68 cm, followed by clove essential oil (mixture 2) with an average inhibition zone of 5.83 cm, and cinnamon essential oil (mixture 1) with an average inhibition zone of 4.98 cm. These results are consistent with the positive effects of components A, B, and C shown in Table 2 . A negative effect was observed for components BC (clove and lemongrass), indicating that the combination of these two oils (mixture 5) reduces the satisfactory inhibitory effect against the bacterium. Figure 2 presents the response surface for the effect of cinnamon, clove, and lemongrass essential oil mixtures on the inhibition of Staphylococcus aureus . Consistent with the results in Table 2 , the negative interaction of BC (clove and lemongrass) decreases the inhibitory efficacy against the bacterium, which is reflected on the surface as areas shifting closer to green tones. Clove, cinnamon, and lemongrass essential oils are widely used in the pharmaceutical and food industries and are known for their strong antibacterial properties, particularly against foodborne pathogens such as Escherichia coli and Staphylococcus aureus (Basak et al., 2021 ; He et al., 2024 ; Kássia da Silva et al., 2024; Silva et al., 2025 ). Since the mathematical models for both bacteria showed low lack of fit, they were combined to determine the best mixture composition for inhibitory activity (Table 3 ). It was confirmed that the optimal condition occurs when 100% lemongrass EO is used. The predicted values were experimentally validated through triplicate analyses, which showed no significant differences at the 95% confidence level. Table 3 Desirability conditions, predicted optimal condition, and predicted and experimentally observed results for the mixture design. Variables and Responses Desirability Criteria for Variables and Concentrations Predicted optimal condition Predicted Values Observed Values Desired Minimum Limit Maximum Limit Importance Escherichia coli Inhibition Zone Maximize 4.1 7.25 3 A (0). B (0) e C (1) 7.20 7.11 ± 0.10 Staphylococcus aureus Inhibition Zone Maximize 3.9 7.25 3 6.63 6.68 ± 0.66 In a study with oregano EO, inhibition zones of 1.7 cm were reported against Staphylococcus aureus , while research with EOs from Lavandula angustifolia Mill., Salvia sclarea L., and Mentha pulegium L. against different Staphylococcus aureus strains showed inhibition zones ranging from 0.6 cm to 2.2 cm (Akhavan et al., 2025 ; Aniba et al., 2025 ). Oregano EO also exhibited an average inhibition zone of 2.73 cm against E. coli , while orange peel EO showed average inhibition zones of 1.5 cm (Ribeiro Cerqueira de Oliveira et al., 2025; Santos et al., 2025 ). These values are lower than those obtained with the essential oils in the present study, indicating that, in addition to exerting stronger effects against the tested bacteria, they also yield better results when combined, compared to other essential oils reported in the literature. 4 Conclusions In the present study, it was observed that for antioxidant activity, clove EO, when used in its pure form (point 2), proved to be the best option for inhibiting DPPH and ABTS radicals. However, binary and ternary combinations of the oils also showed excellent results at the concentration studied. For the inhibition of Botrytis cinerea, the best EO was lemongrass, followed by pure cinnamon and clove oils. The tested combinations of the three oils did not improve the inhibitory effect. For Escherichia coli and Staphylococcus aureus, the strongest inhibitory activity was achieved with 100% lemongrass EO, although the use of alternative mixtures also resulted in significant inhibition (with inhibition zones greater than 6 cm). The use of essential oil mixtures influenced both antioxidant and antimicrobial activities, either through synergistic or antagonistic effects, or simply by diluting the constituents of one EO with another. This remains a topic that requires further research. The different combinations studied here are of great importance for the incorporation of EOs into foods or pharmaceuticals, offering versatility in formulation depending on cost, commercial availability, or desired sensory outcomes. Declarations Competing interests statement The authors declare that they have no competing financial interests or known personal relationships that could have influenced the work reported in this article. Funding This study was funded by the Coordination for the Improvement of Higher Education Personnel—CAPES (code 001). Author Contribution Thamyres César de Albuquerque Sousa: Conceptualization, Methodology, Writing, preparation of the original draft. Thaís Regina Rodrigues Vieira: Conceptualization, Methodology. Glória Caroline Paz Gonçalves: Conceptualization, Methodology. Eliezer Avila Gandra: Visualization, Supervision. Adriana Dillenburg Meinhart: Visualization, Supervision. Acknowledgement We thank the Coordination for the Improvement of Higher Education Personnel (CAPES, Finance Code 001), the National Council for Scientific and Technological Development (CNPq), and the Research Support Foundation of the State of Rio Grande do Sul (FAPERGS). Data Availability All relevant data are included in the manuscript and are available from the corresponding author upon request. References Akakpo, A. Y., Kabore, D., Somda, M. K., Sawadogo, A., Mihin, H. B., Semde, Z., & Ouattara, A. S. 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1","display":"","copyAsset":false,"role":"figure","size":71588,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface plot of the mixture design for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8041198/v1/0f4ea939b1b8c993e9ae701c.jpg"},{"id":96210034,"identity":"bc82145f-1eae-438f-8695-c117076fa6b4","added_by":"auto","created_at":"2025-11-18 18:22:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64185,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface plot of the mixture design for \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8041198/v1/834b58f4e09c49c4b3dcaf10.jpg"},{"id":97892691,"identity":"fa6091af-96fa-46d7-a15e-e5404aef6511","added_by":"auto","created_at":"2025-12-10 15:18:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":955889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8041198/v1/4201cc19-9045-4c22-9a72-821234ff5849.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multivariate Study of the Effects of Combined Cinnamon, Clove, and Lemongrass Essential Oils on Enhancing Antioxidant and Antimicrobial Activity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEssential oils (EOs) are natural substances produced by plants that exhibit a wide range of bioactivities, including antioxidant, antimicrobial, antitumor, anti-acetylcholinesterase, and anti-α-glucosidase activities, among others. These biological properties are mainly attributed to the presence of terpenes, terpenoids, phenols, aldehydes, ethers, and other organic compounds (Krishnan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Niu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Caputo et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; You et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). EOs have been increasingly produced for use in aromatherapy, culinary applications, as food additives, flavor enhancers, fragrances, and other anthropogenic purposes (Sharifi-Rad et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLemongrass EO (\u003cem\u003eCymbopogon citratus\u003c/em\u003e) exhibits antioxidant, antifungal, and antimicrobial activities, making it highly promising for applications aimed at extending food shelf life (Akakpo et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). When applied to tomatoes stored at room temperature (25\u0026deg;C) and under refrigeration (8\u0026deg;C) infected with \u003cem\u003eColletotrichum gloeosporioides\u003c/em\u003e, lemongrass EO reduced the incidence (up to 63%) and severity (up to 9%) of anthracnose at room temperature (within 7 days) and completely inhibited the disease (0% incidence and severity) under refrigeration (within 40 days (Flores \u0026amp; Poveda, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eClove EO (\u003cem\u003eSyzygium aromaticum\u003c/em\u003e) has also demonstrated antioxidant and antimicrobial properties in various studies, proving effective and easy to apply (Pandey et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Suttiarporn et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cinnamon EO is widely used in the food flavoring and cosmetics industries and has shown significant inhibitory effects on most foodborne microorganisms. As a preservative, it has been reported to delay the spoilage of a wide range of foods such as lemons, bread, strawberries, and cheeses (Zhang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In a study conducted by Albuquerque and collaborators, clove, cinnamon, and lemongrass EOs exhibited strong antifungal activity against \u003cem\u003eBotrytis cinerea\u003c/em\u003e, with minimum inhibitory concentrations of 0.85 mg mL⁻\u0026sup1;, highlighting their potential to combat this pathogen (Albuquerque Sousa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eBotrytis cinerea\u003c/em\u003e, he causal agent of gray mold, is one of the most notorious pathogens affecting fruits such as strawberries, leading to severe economic losses both in field production and during the postharvest period. Its impact is largely due to its distinct characteristics, including a short life cycle, massive reproductive capacity, and high genetic variability (Yousef et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This fungus is considered a major contributor to crop losses, with estimates indicating that it is responsible for at least 30% of global production loss annually. It can infect more than 1,400 plant species across nearly 600 genera worldwide, resulting in staggering annual production losses estimated between USD 10 and 100\u0026nbsp;billion (Ullah et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Essential oils play an important role in ensuring food safety against microorganisms such as fungi and bacteria, in addition to extending shelf life through their strong antioxidant potential.\u003c/p\u003e\u003cp\u003eFood safety is a critical concern for public health, as foodborne diseases affect millions of people worldwide, causing significant economic losses and various health problems (Anamul Hasan Chowdhury et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Among the pathogens most commonly associated with foodborne illnesses, Escherichia coli and Staphylococcus aureus are among the most prevalent microorganisms. Escherichia coli is a pathogenic microorganism that contaminates food globally, contributing to 600\u0026nbsp;million foodborne illnesses and 420,000 deaths annually resulting from the consumption of contaminated food (Jangid et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its main symptoms in humans include stomach discomfort, diarrhea, and vomitin (Amente et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a bacterium that produces staphylococcal enterotoxins, whose presence in food can cause outbreaks of food poisoning. This bacterium is part of the microbiota of animals and can spread to food products, with humans also serving as reservoirs (Duchez et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). According to statistics from the National Healthcare Safety Network (NHSN) surveillance system and the Emerging Infections Program (EPS), Staphylococcus aureus infections have been associated with approximately 50,000 annual deaths in the United States. Outbreaks have also been reported in South Korea; between 2002 and 2012, 2,357 cases from 174 outbreaks of staphylococcal food poisoning were documented (Kim et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral studies have demonstrated the effectiveness of EOs when applied individually. Research with lemon leaf EO (\u003cem\u003eCitrus limon\u003c/em\u003e) revealed antioxidant activity against the DPPH radical, with an IC50 of 10.63 ppm, and a minimum inhibitory concentration against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e of 7.8 \u0026micro;g mL⁻\u0026sup1;. Willow leaf EO (\u003cem\u003eHygrophila salicifolia\u003c/em\u003e) exhibited antioxidant activity against the DPPH radical at 2.13 mg mL⁻\u0026sup1; (Xu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yeasmin et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Multiple studies have shown that EOs possess antifungal effects, including activity against the pathogen \u003cem\u003eB. cinerea\u003c/em\u003e. EO from \u003cem\u003eSalvia officinalis\u003c/em\u003e (Rguez et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and EO from \u003cem\u003eCupressus sempervirens\u003c/em\u003e (Rguez et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) demonstrated efficient in vitro antifungal activity against \u003cem\u003eBotrytis cinerea\u003c/em\u003e, while EO from \u003cem\u003eTetraclinis articulata\u003c/em\u003e, applied at a dose of 100 \u0026micro;g mL⁻\u0026sup1; on tomato fruits, reduced infection caused by \u003cem\u003eBotrytis cinerea\u003c/em\u003e by 64.01%(Rguez et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSynergism or antagonism between bioactive compounds remains a developing topic in research (Arora et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Clove and lemongrass essential oils, when used in combination, demonstrated reduced minimum inhibitory concentration (MIC) values against the fungal phytopathogen \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e, exhibiting synergism (Sharma et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, scientific evidence regarding the synergistic or antagonistic effects of lemongrass, cinnamon, and clove essential oils on antioxidant, antimicrobial, and antifungal activities (particularly the inhibition of \u003cem\u003eBotrytis cinerea\u003c/em\u003e) is still limited to our knowledge. If the concentrations of each oil are optimized, the use of essential oil blends may reduce the required amounts of active compounds, enhancing their effects with lower concentrations of each oil while minimizing potential sensory and economic impacts.\u003c/p\u003e\u003cp\u003eIn this context, a multivariate mixture design is a straightforward statistical approach to efficiently optimize combinations, reducing the number of experiments while obtaining high-quality information. This method makes it possible to estimate interactions among variables and to construct predictive models that relate responses to variables (Zonfrillo et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Identifying optimal experimental conditions is a complex task, and multivariate optimization is essential to establish mathematical models capable of predicting the best conditions that meet the selected responses (Effting et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, the aim of this study was to employ a multivariate mixture optimization of lemongrass, clove, and cinnamon essential oils to investigate the interaction effects among these oils on antioxidant activity against DPPH and ABTS radicals and on antimicrobial activity against \u003cem\u003eBotrytis cinerea\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus.\u003c/em\u003e\u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Material and reagents\u003c/h2\u003e\u003cp\u003eThe plant material for the extraction of cinnamon and clove essential oils was purchased from local commerce in Pelotas, Rio Grande do Sul, Brazil (coordinates 31\u0026deg; 46\u0026prime; 19\u0026Prime; S, 52\u0026deg; 20\u0026prime; 34\u0026Prime; O), while the plant material for the extraction of lemongrass essential oil was obtained from a rural producer in Cap\u0026atilde;o do Le\u0026atilde;o, Rio Grande do Sul, Brazil (coordinates 31\u0026deg;45'48\"S e 52\u0026deg;29'02\"O). The culture media used were potato dextrose agar (PDA), obtained from KASVI (Spain); tryptic soy broth (TSB), KASVI (Spain); eosin methylene blue agar (EMB), KASVI (Spain); Baird-Parker agar, NutriSelect Plus (Germany); and Mueller-Hinton agar, KASVI (Spain). 2,2-diphenyl-1-picrylhydrazyl (DPPH), CAS 1898-66-4, and 2,2\u0026prime;-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), CAS 30931-67-0, were purchased from Sigma-Aldrich (Germany). Methanol PA, CAS 67-56-1, and potassium persulfate, CAS 7727-21-1, were obtained from \u0026Ecirc;xodo Cient\u0026iacute;fica (Brazil). The fungi were isolated from strawberry plants (\u003cem\u003eFragaria \u0026times; ananassa\u003c/em\u003e Duch.) cultivated in the region of Pelotas, Brazil, and identified by their morphological characteristics (grayish growth, branched hyphae and conidiophores, unicellular ovoid conidia, colorless or grayish at the apex of conidiophores) as \u003cem\u003eBotrytis cinerea\u003c/em\u003e. Standard bacterial strains of \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 43895) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e were used (ATCC 10832).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Extraction of essential oils\u003c/h2\u003e\u003cp\u003eThe extraction of essential oils was carried out by hydrodistillation using a fixed Clevenger apparatus, according to the method described by Albuquerque Sousa et al., (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e1. Multivariate mixture design\u003c/h3\u003e\n\u003cp\u003eThe design employed was of the Simplex Centroid type, as described by Neto (2010). In this system, the experimental points are symmetrically distributed, making it possible to study the individual effects of the oils, as well as the binary and ternary combination effects. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the variables and levels studied, which resulted in the evaluation of 10 mixture combinations, each performed in triplicate and in random order.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u0026ndash; Variables, levels, and responses of the multivariate mixture design\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMixture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eComposition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eAntioxidant activity (% inhibition)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003eAntimicrobial activity\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCinnamon\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClove\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLemongrass\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDPPH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eABTS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eB. cinerea\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(% inhibition)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eE. coli (cm of halo)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eS. aureus (cm of halo)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e37.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e76.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e46.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e79.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e47.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e79.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e45.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e86.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e70.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e86.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e71.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e30.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e86.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e71.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e29.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e94.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e82.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e94.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e83.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e33.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e95.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e85.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e63.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e45.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e92.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e64.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e45.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e62.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e42.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eHigher values are represented in the green scale, intermediate values in yellow, and lower values in the red scale\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor the analyses of antioxidant activity and antifungal effect against \u003cem\u003eBotrytis cinerea\u003c/em\u003e, the three pure EOs were diluted to a concentration of 0.08 mg mL⁻\u0026sup1; in 1X phosphate-buffered saline (PBS, pH 7.4). A total of 500 mL of PBS solution was prepared containing 4 g of sodium chloride, 0.1 g of potassium chloride, 0.72 g of disodium phosphate, and 0.1225 g of monopotassium phosphate in distilled water. This concentration was defined based on a previous study by the research group, which demonstrated inhibitory effects against the fungus(Albuquerque Sousa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). hus, the mixtures were prepared with the following composition (in mg/mL): 1 \u0026ndash; Cinnamon EO 0.08; 2 \u0026ndash; Clove EO 0.08; 3 \u0026ndash; Lemongrass EO 0.08; 4 \u0026ndash; Cinnamon EO 0.04\u0026thinsp;+\u0026thinsp;Clove EO 0.04; 5 \u0026ndash; Clove EO 0.04\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.04; 6 \u0026ndash; Cinnamon EO 0.04\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.04; 7 \u0026ndash; Cinnamon EO 0.0267\u0026thinsp;+\u0026thinsp;Clove EO 0.0267\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.0266; 8 \u0026ndash; Cinnamon EO 0.053\u0026thinsp;+\u0026thinsp;Clove EO 0.0133\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.0133; 9 \u0026ndash; Cinnamon EO 0.0133\u0026thinsp;+\u0026thinsp;Clove EO 0.053\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.0133; 10 \u0026ndash; Cinnamon EO 0.0133\u0026thinsp;+\u0026thinsp;Clove EO 0.0133\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.053.\u003c/p\u003e\u003cp\u003eFor the analyses of antibacterial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, each pure EO was diluted to a concentration of 1 mg mL⁻\u0026sup1;. Thus, the mixtures had the following composition (in mg/mL): 1 \u0026ndash; Cinnamon EO 1.00; 2 \u0026ndash; Clove EO 1.00; 3 \u0026ndash; Lemongrass EO 1.00; 4 \u0026ndash; Cinnamon EO 0.50\u0026thinsp;+\u0026thinsp;Clove EO 0.50; 5 \u0026ndash; Clove EO 0.50\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.50; 6 \u0026ndash; Cinnamon EO 0.50\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.50; 7 \u0026ndash; Cinnamon EO 0.34\u0026thinsp;+\u0026thinsp;Clove EO 0.33\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.34; 8 \u0026ndash; Cinnamon EO 0.67\u0026thinsp;+\u0026thinsp;Clove EO 0.16\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.16; 9 \u0026ndash; Cinnamon EO 0.16\u0026thinsp;+\u0026thinsp;Clove EO 0.67\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.16; 10 \u0026ndash; Cinnamon EO 0.16\u0026thinsp;+\u0026thinsp;Clove EO 0.16\u0026thinsp;+\u0026thinsp;Lemongrass EO 0.67.\u003c/p\u003e\u003cp\u003eThe responses studied were antioxidant activity (measured by the percentage of inhibition of DPPH and ABTS radicals) and antimicrobial activity (measured by the inhibition zones of \u003cem\u003eBotrytis cinerea\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains).\u003c/p\u003e\u003cp\u003eSubsequently, the responses obtained were evaluated for the possibility of developing mathematical models with adequate fit to explain the interaction effects and the behavior of EO mixtures in relation to the responses, as well as to predict new mixture combinations and their effects. The models were analyzed by means of Analysis of Variance (ANOVA) using Statistica 12 software (StatSoft, USA), with a 95% confidence level.\u003c/p\u003e\n\u003ch3\u003e2. Methods for antioxidant and antifungal activity analysis\u003c/h3\u003e\n\u003cp\u003eThe antioxidant capacity of the microparticles was evaluated based on their ability to donate hydrogen to the stable free radical DPPH (Rufino et al., 2007a) and to scavenge the free radical ABTS (Rufino et al., 2007b), with minor modifications. For the DPPH assay, 100 \u0026micro;L of the EO dilutions were added to 3.9 mL of a 0.06 mM methanolic DPPH solution. The reaction was carried out at 25\u0026deg;C, in the absence of light, for 1 h, and absorbance was measured at 515 nm. For the ABTS assay, the free radical was prepared by reacting 5.7 mL of a 7 mM ABTS solution with 0.1 mL of a 140 mmol/L potassium persulfate solution at 25\u0026deg;C, under low light, for 16 h. Then, 30 \u0026micro;L of the EO dilutions were added to 3 mL of ABTS solution, and absorbance was measured at 734 nm. The results were expressed as percentage inhibition according to the equation below:\u003c/p\u003e\u003cp\u003eInhibition (%) = (A \u003csub\u003econtrol\u003c/sub\u003e \u0026minus; A \u003csub\u003eamostra\u003c/sub\u003e )/A \u003csub\u003econtrol\u003c/sub\u003e * 100, where A represents absorbance.\u003c/p\u003e\u003cp\u003eThe antifungal activity against \u003cem\u003eBotrytis cinerea\u003c/em\u003e was determined using the method described by Gakuubi et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), with some modifications. Initially, \u003cem\u003eBotrytis cinerea\u003c/em\u003e was pre-cultivated at 25\u0026deg;C for 72 h on potato dextrose agar (PDA). Approximately 25 mL of sterile PDA were prepared and poured into sterile Petri dishes at a temperature below 50\u0026deg;C. Immediately afterward, the EO mixtures were added and homogenized in the still-liquid PDA medium. A well of approximately 6 mm in diameter was made at the center of the plate, where a PDA disk containing the cultivated fungus (also 6 mm in diameter) was placed. The plates were then sealed and incubated at 25\u0026deg;C for 96 h. The growth halos were measured daily during incubation to calculate the percentage of inhibition. A plate without the addition of EO mixtures was used as the control.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Analysis of antibacterial activity.\u003c/h2\u003e\u003cp\u003eThe antibacterial analysis against \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e was carried out according to the protocol proposed by the Clinical and Laboratory Standards Institute Manual \u0026ndash; CLSI (CLSI, 2005), with modifications. For reactivation, an inoculum of each bacterium was transferred to tryptic soy broth (TSB) and incubated in an oven for 24 h at 37\u0026deg;C. Subsequently, an inoculum from this growth was streaked onto Petri dishes with selective media, eosin methylene blue agar (EMB) for \u003cem\u003eEscherichia coli\u003c/em\u003e and Baird-Parker agar for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and incubated for 24 h at 37\u0026deg;C to isolate the colonies.\u003c/p\u003e\u003cp\u003eFrom the bacterial growth on Petri dishes, an inoculum was taken and resuspended in saline solution (0.85% NaCl), which was standardized to a 0.5 concentration on the McFarland scale (1.5 \u0026times; 10⁸ CFU mL⁻\u0026sup1;). For the well diffusion test, the saline solution containing the bacterial inoculum was spread with the aid of a sterile swab onto the surface of Muller-Hinton agar plates (for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e) and EMB agar plates (for \u003cem\u003eEscherichia coli\u003c/em\u003e), where wells had been previously made. Then, 10 \u0026micro;L of the mixtures were placed into the wells, and the plates were incubated for 24 h at 37\u0026deg;C. After this period, the inhibition zones were measured, and the results were expressed in centimeters.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results and discussions","content":"\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the results obtained for antioxidant, antifungal, and antibacterial activities provided by the mixtures of the experimental design. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the validation of the mathematical models regarding lack of fit, significance of the regressions, and significant coefficients.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eValidation of the mathematical models of the essential oil mixture design\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eResponses\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eModel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e\u003cp\u003eSignificant Model Coefficients (error)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF Lack of Fit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF critical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF Model Significance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF critical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eABC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e% DPPH Inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecial Cubic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e162.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e178.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e733.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e150.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e5.18\u003c/sub\u003e= 2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e27.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e6.23\u003c/sub\u003e = 2.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(8.86)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(44.53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(44.53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(294.31)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e% ABTS Inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecial Cubic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e89.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e80.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e61.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e771.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e78.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e5.18\u003c/sub\u003e = 2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e52.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e6.23\u003c/sub\u003e = 2.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(5.05)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(5.05)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(5.05)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(25.36)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(25.36)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(167.61)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e% \u003cem\u003eBotrytis\u003c/em\u003e Inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eQuadratic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e68.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-83.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-73.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e51.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e6.18\u003c/sub\u003e= 2.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e15.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e5.24\u003c/sub\u003e = 2.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(3.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(3.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(3.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(14.34)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(14.34)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInhibition zone\u003c/p\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSpecial Cubic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-3.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e21.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e7.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e5.18\u003c/sub\u003e = 2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e7.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e6.23\u003c/sub\u003e = 2.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(0.32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(0.32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(0.32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e(1.63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e(1.63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e(10.75)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInhibition zone\u003c/p\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eQuadratic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-3.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e6.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e6.18\u003c/sub\u003e = 2.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e4.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eF\u003csub\u003e5.24\u003c/sub\u003e = 2.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(0.37)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(0.37)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e(0.37)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e(1.71)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"13\"\u003eA: Cinnamon essential oil. B: Clove essential oil. C: Lemongrass essential oil;\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Antioxidant activity\u003c/h2\u003e\u003cp\u003eFor antioxidant activity, measured through the inhibition of DPPH and ABTS radicals, the model that best explained the behavior of the variables was the special cubic model. However, in the lack-of-fit test, it was observed that for DPPH the calculated F value was 150.70, far exceeding the F\u003csub\u003ecalculate\u003c/sub\u003e value of 5.18 and 2.77. This high lack of fit makes the use of this mathematical model unfeasible for predicting optimal conditions. The same occurred for the ABTS model, with a calculated F value of 78.98, also exceeding the F\u003csub\u003ecalculate\u003c/sub\u003e value of 5.18 and 2.77. Therefore, the interpretation of the observed effects for these responses was based on the experimental results (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhen the essential oils were studied individually (mixtures 1, 2, and 3), the best antioxidant activity was observed in clove oil (mixture 2), with 94.97% inhibition for DPPH and 93.85% for ABTS. Pure cinnamon and lemongrass essential oils exhibited low antioxidant activity (below 9% for DPPH and 17% for ABTS).\u003c/p\u003e\u003cp\u003eFor the binary mixtures, whenever clove oil was present, inhibition was greater than 90% for DPPH and greater than 66% for ABTS, regardless of whether it was combined with cinnamon or lemongrass oil (mixtures 4 and 5). In the absence of clove (binary mixture of cinnamon and lemongrass), inhibition was lower than 18% for both radicals.\u003c/p\u003e\u003cp\u003eFor the ternary mixtures (mixtures 7, 8, 9, and 10), inhibition of the DPPH radical was greater than 86%, and inhibition of the ABTS radical was greater than 62%. The highest inhibition for the ABTS radical was observed in mixture 9, with values above 82%, which contained a higher proportion of clove essential oil. The presence of clove oil consistently resulted in the best antioxidant activity.\u003c/p\u003e\u003cp\u003eWhen clove EO accounted for one-third of the mixture (mixture 7), the antioxidant activity achieved was above 90% for DPPH and above 76% for ABTS. This indicates the possibility of varying the combinations used depending on cost, commercial availability, and sensory compatibility.\u003c/p\u003e\u003cp\u003eThe biological activity of clove essential oil results from its high content of bioactive compounds such as eugenol, which contains a phenolic group of interest due to its strong antioxidant capacity, as well as antimicrobial activity, including action against foodborne pathogenic bacteria (El-Saber Batiha et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Haro-Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khalil et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ulanowska \u0026amp; Olas, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Citral, the main constituent of lemongrass essential oil, is known for its anti-inflammatory, immunomodulatory, fungistatic antimicrobial, antioxidant, and antiseptic properties (Alagawany et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cinnamon essential oil has cinnamaldehyde as its major compound and is widely used in the pharmaceutical and food industries for its strong antioxidant and antifungal properties (Benmoussa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRegarding antioxidant activity, it can be observed that although the models showed a high lack of fit, the significant coefficients were consistent with the experimental results, including their numerical magnitude. One possible reason for a false outcome in the lack-of-fit test is when the relative standard deviation of the replicates is very low. However, the deviation in this study was within normal limits and did not affect the test. The levels were selected to allow testing of several combinations in different proportions. The lack of fit is possibly due to the absence of terms in the model to account for the variance (which would require a larger number of experiments) or to the range between the levels studied.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Antifungal activity\u003c/h2\u003e\u003cp\u003eFor the activity against the fungus \u003cem\u003eBotrytis cinerea\u003c/em\u003e, the model that best explained the behavior of the variables was the quadratic model. However, in the lack-of-fit test, the F\u003csub\u003ecalculated\u003c/sub\u003e value was 51.20, which was much higher than the F\u003csub\u003ecritical 6,18\u003c/sub\u003e of 2,66.\u003c/p\u003e\u003cp\u003eWhen interpreting the effects observed from the experimental results (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), it can be noted that when the essential oils were tested individually (mixtures 1, 2, and 3), the strongest inhibitory effect against the fungus was obtained with lemongrass essential oil, with values above 67%, followed by cinnamon oil with values above 49% and clove oil with values above 44%. For the binary and ternary mixtures (mixtures 4, 5, 6, 7, 8, 9, and 10), all showed inhibition values lower than those of the oils when used alone, indicating that the oil mixtures did not produce a synergistic effect but rather an antagonistic one, reducing the inhibitory effect against the fungus due to the lower proportion of clove oil. Despite the lack of fit in the quadratic model, the coefficients also pointed to similar responses. The most significant mathematical effects were the negative interaction effects (AC and BC), namely between cinnamon essential oil (A) and lemongrass (C), and between clove essential oil (B) and lemongrass. The model also confirmed the experimental data, highlighting that the third most important effect was the positive contribution of lemongrass to the inhibition percentage when this EO was used individually (C).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Antibacterial activity\u003c/h2\u003e\u003cp\u003eFor the antibacterial activity against the pathogens \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, the model that best explained the behavior of the variables was the special cubic model for \u003cem\u003eEscherichia coli\u003c/em\u003e and the quadratic model for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. In the lack-of-fit test, the F \u003csub\u003ecalculated\u003c/sub\u003e value for \u003cem\u003eEscherichia coli\u003c/em\u003e was 7.25, slightly higher than the F \u003csub\u003ecritical\u003c/sub\u003e value of 5.18 and 2.77. For the \u003cem\u003eStaphylococcus aureus\u003c/em\u003e model, the F\u003csub\u003ecalculated\u003c/sub\u003e value was 6.91, slightly higher than the F \u003csub\u003ecritical 6.18\u003c/sub\u003e value and 2.66. Both models showed a slight lack of fit. However, since these values were less than 10 times the F \u003csub\u003ecritical\u003c/sub\u003e value, several researchers have successfully employed them to predict optimal conditions. Therefore, both models were used for this purpose (Caroline Paz Gon\u0026ccedil;alves et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rosas et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe significant coefficients presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e are consistent with the values observed in the experiment (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For Escherichia coli, pure lemongrass EO (coefficient C) showed the largest inhibition zone (greater than 7 cm), corroborating the experimental results reported in Section 3. The next most significant effect was observed with cinnamon EO, followed by clove EO, both of which also had significant coefficients. Regarding binary effects, coefficient AB (relating cinnamon and clove, mixture 4 in the experiment) showed a positive interaction in terms of bacterial inhibition. On the other hand, coefficient AC (mixture 6) showed a negative interaction between cinnamon and lemongrass oils.\u003c/p\u003e\u003cp\u003eThe simultaneous use of the three EOs also showed a positive effect. When lemongrass EO was present in the mixture of the three EOs at a proportion of two-thirds (mixture 10), the inhibition zone was greater than 6 cm, a value higher than that obtained with cinnamon or clove oils when used alone. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the response surface for the effect of the mixtures of clove, cinnamon, and lemongrass essential oils on the inhibition of Escherichia coli, showing that higher concentrations of lemongrass maximize inhibition, while mixtures with a predominant proportion of this oil also maintain high efficacy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the inhibition of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, the best antibacterial activity was observed with lemongrass essential oil (mixture 3), with an average inhibition zone of 6.68 cm, followed by clove essential oil (mixture 2) with an average inhibition zone of 5.83 cm, and cinnamon essential oil (mixture 1) with an average inhibition zone of 4.98 cm. These results are consistent with the positive effects of components A, B, and C shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A negative effect was observed for components BC (clove and lemongrass), indicating that the combination of these two oils (mixture 5) reduces the satisfactory inhibitory effect against the bacterium. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents the response surface for the effect of cinnamon, clove, and lemongrass essential oil mixtures on the inhibition of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Consistent with the results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the negative interaction of BC (clove and lemongrass) decreases the inhibitory efficacy against the bacterium, which is reflected on the surface as areas shifting closer to green tones.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eClove, cinnamon, and lemongrass essential oils are widely used in the pharmaceutical and food industries and are known for their strong antibacterial properties, particularly against foodborne pathogens such as \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Basak et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; He et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; K\u0026aacute;ssia da Silva et al., 2024; Silva et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSince the mathematical models for both bacteria showed low lack of fit, they were combined to determine the best mixture composition for inhibitory activity (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It was confirmed that the optimal condition occurs when 100% lemongrass EO is used. The predicted values were experimentally validated through triplicate analyses, which showed no significant differences at the 95% confidence level.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDesirability conditions, predicted optimal condition, and predicted and experimentally observed results for the mixture design.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables and Responses\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eDesirability Criteria for Variables and Concentrations\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePredicted optimal condition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePredicted Values\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eObserved Values\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDesired\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMinimum Limit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMaximum Limit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eImportance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\u003cp\u003eInhibition Zone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMaximize\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eA (0). B (0) e C (1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e Inhibition Zone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMaximize\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn a study with oregano EO, inhibition zones of 1.7 cm were reported against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, while research with EOs from \u003cem\u003eLavandula angustifolia\u003c/em\u003e Mill., \u003cem\u003eSalvia sclarea\u003c/em\u003e L., and \u003cem\u003eMentha pulegium\u003c/em\u003e L. against different \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains showed inhibition zones ranging from 0.6 cm to 2.2 cm (Akhavan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Aniba et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Oregano EO also exhibited an average inhibition zone of 2.73 cm against \u003cem\u003eE. coli\u003c/em\u003e, while orange peel EO showed average inhibition zones of 1.5 cm (Ribeiro Cerqueira de Oliveira et al., 2025; Santos et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These values are lower than those obtained with the essential oils in the present study, indicating that, in addition to exerting stronger effects against the tested bacteria, they also yield better results when combined, compared to other essential oils reported in the literature.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn the present study, it was observed that for antioxidant activity, clove EO, when used in its pure form (point 2), proved to be the best option for inhibiting DPPH and ABTS radicals. However, binary and ternary combinations of the oils also showed excellent results at the concentration studied. For the inhibition of Botrytis cinerea, the best EO was lemongrass, followed by pure cinnamon and clove oils. The tested combinations of the three oils did not improve the inhibitory effect. For Escherichia coli and Staphylococcus aureus, the strongest inhibitory activity was achieved with 100% lemongrass EO, although the use of alternative mixtures also resulted in significant inhibition (with inhibition zones greater than 6 cm). The use of essential oil mixtures influenced both antioxidant and antimicrobial activities, either through synergistic or antagonistic effects, or simply by diluting the constituents of one EO with another. This remains a topic that requires further research. The different combinations studied here are of great importance for the incorporation of EOs into foods or pharmaceuticals, offering versatility in formulation depending on cost, commercial availability, or desired sensory outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests statement\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing financial interests or known personal relationships that could have influenced the work reported in this article.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was funded by the Coordination for the Improvement of Higher Education Personnel\u0026mdash;CAPES (code 001).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThamyres C\u0026eacute;sar de Albuquerque Sousa: Conceptualization, Methodology, Writing, preparation of the original draft. Tha\u0026iacute;s Regina Rodrigues Vieira: Conceptualization, Methodology. Gl\u0026oacute;ria Caroline Paz Gon\u0026ccedil;alves: Conceptualization, Methodology. Eliezer Avila Gandra: Visualization, Supervision. Adriana Dillenburg Meinhart: Visualization, Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the Coordination for the Improvement of Higher Education Personnel (CAPES, Finance Code 001), the National Council for Scientific and Technological Development (CNPq), and the Research Support Foundation of the State of Rio Grande do Sul (FAPERGS).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll relevant data are included in the manuscript and are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkakpo, A. Y., Kabore, D., Somda, M. K., Sawadogo, A., Mihin, H. B., Semde, Z., \u0026amp; Ouattara, A. S. (2023). 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Encapsulation and delivery systems of cinnamon essential oil for food preservation applications. \u003cem\u003eAdvances in Colloid and Interface Science\u003c/em\u003e, \u003cem\u003e318\u003c/em\u003e, 102965. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cis.2023.102965\u003c/span\u003e\u003cspan address=\"10.1016/j.cis.2023.102965\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZonfrillo, B., Bellumori, M., Digiglio, I., Innocenti, M., Orlandini, S., Furlanetto, S., Khatib, M., Papini, A., Mainente, F., Zoccatelli, G., \u0026amp; Mulinacci, N. (2025). Multivariate optimization of ulvan extraction applying Response Surface Methodology (RSM): the case of Ulva lactuca L. from Orbetello lagoon. \u003cem\u003eCarbohydrate Polymers\u003c/em\u003e, \u003cem\u003e354\u003c/em\u003e, 123340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2025.123340\u003c/span\u003e\u003cspan address=\"10.1016/j.carbpol.2025.123340\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Botrytis cinerea, Bioactive Compounds, Escherichia coli, Mixture Design, Staphylococcus aureus","lastPublishedDoi":"10.21203/rs.3.rs-8041198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8041198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA multivariate mixture system of EOs was employed to evaluate individual, binary, and tertiary effects of cinnamon, clove, and lemongrass essential oils on in vitro antioxidant activity and antimicrobial activity against \u003cem\u003eBotrytis cinerea\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. The highest antioxidant activity was observed with 100% clove oil, showing inhibition rates of 94.97% (DPPH), 93.85% (ABTS). The addition of clove EO to the mixture, in a two-thirds proportion, resulted in inhibition percentages above 80% for both DPPH and ABTS. Regarding the inhibitory effect \u003cem\u003eBotrytis cinerea\u003c/em\u003e, lemongrass EO exhibited the best performance, with 68% inhibition, while its combination, even partially, with clove and cinnamon produced a negative effect. \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, the strongest inhibitory activity was obtained with 100% lemongrass EO (inhibition zones of 7.1 and 6.7 cm). The simultaneous use lemongrass and cinnamon EOs in higher proportions within the mixture resulted in greater inhibition, suggesting a synergistic effect.\u003c/p\u003e","manuscriptTitle":"Multivariate Study of the Effects of Combined Cinnamon, Clove, and Lemongrass Essential Oils on Enhancing Antioxidant and Antimicrobial Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 18:22:43","doi":"10.21203/rs.3.rs-8041198/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"602da76a-27ec-42fc-831a-b42c12898579","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T11:09:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 18:22:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8041198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8041198","identity":"rs-8041198","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}